Abstract: Dagger and needle nematodes are considered globally one of the 10 mosteconomically important nematode groups. They interact with the plants by direct damage infeeding process from them or in plant virus transmission. The accurate and timely identificationof dagger and needle nematodes infecting vineyards is a prerequisite for designing effectivemanagement strategies. This is particularly relevant in grapevine fields because of the diversity ofthese nematodes and the need for distinction between virus vector and non-virus vectornematodes. This present study establishes the biodiversity of longidorids found in grapevines atsouthern Spain, as well as their integrative diagnosis and molecular phylogenetic relationships,using morphometrical analysis and molecular markers including D2-D3 expansion regions of 28SrRNA, 18S rRNA and ITS1-rRNA. Xiphinema pachtaicum and X. index were the most frequentlydagger nematodes found (90.8 and 30.3% frequency of infestation, respectively), but otherdagger species included X. adenohystherum, X. hispidum, X. italiae, X. lupini, X. nuragicum,X. rivesi, and X. turcicum (1.3%, 7.9%, 13.2%, 3.9%, 1.3%, 2.6%, and 1.3% frequency ofinfestation, respectively). Needle nematodes were less abundant and diverse including onlyLongidorus alvegus and L. magnus (2.6% and 11.8% frequency of infestation, respectively). Thespecies identification based on sequencing of rRNA regions and BLAST analysis was congruentwith species identification based on morphometrical studies. D2-D3 expansion segments of 28SrDNA and ITS1 were most useful for dagger and needle nematode species identification sincethey showed more variability than partial 18S rDNA. Results also showed a high level ofnematode endemism (such as X. adenohystherum, X. hispidum, X. nuragicum, X. lupini andL. alvegus) and their phylogenetic grouping suggest a common origin for several of thelongidorid species found and the Iberian Peninsula as their potential centre of origin. Phylogenyshowed two major clades well defined and supported including Longidorus and Xiphinemaspecies. However, the grouping of Longidorus and Paralongidorus species with the rest ofspecies is well supported. Nevertheless, tree topology analysis by Shimodaira-Hasegawa test ofD2-D3 and partial 18S of our broad sequences did not refute the monophyly of the genusXiphinema, but the genus Paralongidorus was not accepted as a valid.